OVERVIEW

We have developed a novel multicomponent biomanufacturing platform, Coup Dy’état, to optimize the bioproduction. We have successfully demonstrated that each module of our system works as intended. On top of that, we have also integrated several modules, and proved that they are able to complement one another.

XYLOSE AS FEEDSTOCK

✔Successfully constructed an inducible xylose-utilizing module

✔Demonstrated improved growth of E. coli BL21* containing this module in xylose and glucose-xylose mixture

Future work aims to produce naringenin using xylose as the feedstock for E. coli.

DE NOVO BIOSYNTHESIS

✔Successfully constructed a naringenin-producing plasmid with just a single missing enzyme required for full de novo synthesis

✔Demonstrated production of naringenin from coumaric acid in E. coli Acella and BL21*

LUTEOLIN

✔Successfully constructed chemically-inducible and light inducible luteolin-producing plasmids

✔Characterized Bba_ (F3’H), Bba (FNS), Bba (pBAD-FNS)

✔Demonstrated production and extraction of luteolin from naringenin in E. coli BL21*

Future work aims to produce luteolin, as well as other flavonoids from de novo naringenin.

BLUE LIGHT REPRESSIBLE SYSTEM

✔Improved characterization of EL222 blue light repressible promoter, P_BLrep (BBa_K2819103)

✔Demonstrated blue light repression of luteolin production

STRESS REPORTER

✔Successfully constructed a stress reporter module

✔Characterized the burden responsive promoter, P_htpG1

✔Demonstrated that reporter is robust under different conditions

✔Demonstrated stress induced by naringenin- and luteolin-producing plasmids

✔Demonstrated that reporter is able to detect stress induced by naringenin- and luteolin-producing plasmids

CELL-MACHINE INTERFACE

✔Designed and built devices which help characterize optogenetic circuits in petri dishes and 250 ml conical flasks

✔Designed and built a 500 ml working volume benchtop optogenetic bioreactor, which comprises a peristaltic pump, 2-in-1 OD and fluorescence sensor, and fermentation chamber

✔Designed and implemented a feedback control system to control the optogenetic bioreactor

✔Demonstrated that 2-in-1 OD and fluorescence is able to measure OD600 and fluorescence

✔Demonstrated that feedback control system is able to turn off blue light in the presence of fluorescence